The present disclosure relates to an image obtaining apparatus, an image obtaining method, and an image obtaining program obtaining an image by using a microscope.
Flow cytometry is known as a method of analyzing and sorting minute particles such as biological tissues. A flow cytometry apparatus (flow cytometer) is capable of obtaining, at high speed, shape information and fluorescence information from each particle such as a cell. The shape information includes size and the like. The fluorescence information is information on DNA/RNA fluorescence stain, and on protein and the like dyed with fluorescence antibody. The flow cytometry apparatus (flow cytometer) is capable of analyzing correlations thereof, and of sorting a target cell group from the particles. Further, imaging cytometry is known as a method of performing cytometry based on a fluorescent image of a cell. In the imaging cytometry, a fluorescent image of a biological sample on a glass slide or a dish is magnified and photographed. Information on each cell in the fluorescent image is digitalized and quantified. The information includes, for example, an intensity (brightness), size, and the like of bright points, which mark a cell. Further, the cell cycle is analyzed, and other processing is performed.
In a case of obtaining a fluorescent image of a biological sample, which is fluorescence-stained, the focus is moved at predetermined distances in the thickness direction of the biological sample. Each of fluorescent images at the plurality of focuses is generated as image data. As a result, all the bright points, which mark a target cell, may be reliably obtained. However, in this case, the number of fluorescent images for one biological sample is extremely large. Further, there is a tendency that processing load and data volume for one biological sample are increased.
If a distance between focus positions is made larger, the number of fluorescent images for one biological sample is decreased. However, in this case, some bright points, which mark target cells, may not be obtained. As a result, analysis accuracy is lowered. Further, the following case is discussed. That is, the focus depth of the objective lens is increased by lowering the NA of an objective lens or by another method. However, in this case, the resolutions of all the fluorescent images are decreased uniformly. As a result, analysis accuracy is lowered after all.
In relation to the above-mentioned circumstances, for example, Japanese Patent Application Laid-open No. 2011-017982 discloses a method of obtaining a fluorescent image as follows. According to this method, a fluorescent-image obtaining unit moves a movable stage in a Z-axis direction (optical-axis direction), and moves the focus of an optical system on a sample site in the thickness direction. The fluorescent-image obtaining unit exposes an image sensor to light from the time point when the movement of the movable stage in the Z-axis direction is started to the time point when the movement of the movable stage is finished. The fluorescent-image obtaining unit obtains a fluorescent image of a sample site from the image sensor. The fluorescent image is an image obtained as the result of the light-exposure and obtained at the final time point.